The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Melanoma, a cancer of the pigment-producing cells in the skin epidermis, can be highly metastatic, and malignant melanomas are relatively resistant to standard chemotherapy. A major cause for melanoma initiation is extensive or intermittent exposure to the sun's radiation over a period of time, and the extent of melanin pigmentation is an important risk factor. The exact molecular mechanisms that lead to melanoma are complex and poorly understood, and may involve both mutagenic DNA lesions and epigenetic misregulation. The complexity is added by the involvement of several different signal transduction pathways, such as the Hedgehog pathway, which controls BCL2-mediated apoptosis; mutations in the Patched gene, the endpoint of the Hedgehog pathway, have also been correlated with skin cancers [3,12-15].
A frequent causative mechanism for an inherited form of predisposition to melanoma is thought to be a chromosomal deletion over 9p21. The 9p21 site harbors the tumor suppressor gene INK4a and accompanies additional inactivating mutations that lead to the constitutive activation of genes such as BRAF [16,17]. INK4a encodes one of several cyclin-dependent protein kinase inhibitors, which is located adjacent to an alternate reading frame of the human p14ARF. P14ARF binds to the Mdm2 protein in several cell lines (though remains untested in melanoma cell lines, to our knowledge) and thereby abrogates Mdm2's binding to p53, causing p53 to be stabilized and nuclear localized. The loss of INK4a therefore may lead to interference of two separate pathways of cell cycle control: CDK signaling and suppression of p53 activity by Mdm2-induced acceleration of p53 degradation. Methylation near the 5′ upstream region of INK4a has been shown in some 10% of melanomas [7], suggesting that epigenetic down-regulation of this gene may be important for melanoma development. The activation of BRAF alone may be insufficient to cause metastatic melanoma, but additional mutagenic or epigenetic events such as the inactivation of tumor suppressor genes, e.g., Pten [18], may be important. There is evidence that the NOTCH signaling pathway is also important for distinguishing normal melanocytes from melanoma cells [19,20]. But, BRAF is mutated in over 60% of human melanoma. Several small-molecule inhibitors of BRAF are known, such as vemurafenib, but a significant hurdle to its use exists due to the emergence of vemurafenib-resistant cells
Measurement of genome-wide DNA copy number variations, together with analysis of somatic mutations in specific marker genes, can be used to distinguish among different melanoma subtypes with reasonable accuracy [21]. Particularly noteworthy is the recent demonstration of abnormally high oncogenic potentials of single melanoma cells [22], emphasizing the need for a better understanding the molecular mechanisms of melanoma progression.
In the search for such an understanding, attention has recently focused on the role of small non-coding RNA molecules in cancer development [23-27] and in melanoma in particular [28-32]. miRNAs influence cancer development by serving either as tumor suppressors or oncogenes [33-39] by their negative regulatory effects on mRNA encoded by oncogenes or tumor suppressor genes, respectively. With the goal of defining the genes with major contributions to melanoma, several genome-wide expression level studies have identified a number of protein-coding [40] and microRNA (miRNA) genes as important players [32,41-43]. Several of these genes and their expression signatures exhibit distinct patterns among malignant metastatic melanomas and their benign forms, but their significance with respect to melanoma initiation and progression is poorly understood. For example, miR-221/222 were found to down-regulate p27Kip1/CDKN1B and the c-KIT receptor, which controls the progression of neoplasia leading to enhanced proliferation and reduced differentiation in melanoma cells [42]. Similarly, high miR-137 expression in melanoma cell lines down-regulates microphthalma associated transcription factor (MITF), a transcription factor important for melanocyte cell growth, maturation, apoptosis, and pigmentation [32]. The depletion of miR-182 reduces invasiveness and induces melanoma cell death by suppressing the expression of transcription factors FOXO3 and MITF [43], suggesting that its increased expression may be associated with certain aspects of melanoma biology.
With such a number of interrelated genetic causes of melanoma, and with the resulting wide array of individual phenotypes that may result from the various permutations, a clear need remains for improved methods of diagnostics of individual resistance to chemotherapeutic agents, as well as methods of treatment in patients exhibiting the same. Furthermore, one hallmark of melanoma is the ability to re-route major pathways of energy provision and consumption to support the energy demands associated with growth and survival; therefore, there remains a need for a more complete understanding of the mechanistic role of microRNAs in modulating target genes that affect protein, carbohydrate, and lipid metabolism, and, therefore, contribute to melanoma.